Poliovirus-specific primer-dependent RNA polymerase able to copy poly(A).

In order to identify the viral polymerase involved in cowpea mosaic virus (CPMV) RNA replication the 87K, ll0K and 170K proteins as well as the complete 200K polyprotein of CPMV B-RNA have been produced in cowpea protoplasts, using expression vectors based on the 35S promoter of cauliflower mosaic virus. CPMVspecific proteins were obtained that were indistinguishable from proteins found in CPMV-infected protoplasts. Proteolytic processing of precursor proteins synthesized from the expression vectors proved that the 24K protease contained within these proteins is active.Moreover, it was established that protoplasts transfected with the expression vector containing the entire 200K coding sequence, but not those transfected with vectors containing the 170K, ll0K or 87K coding sequences, were able to support replication of co-inoculated M-RNA. Despite the ability to support replication of M-RNA for protoplasts transiently expressing the 200K coding region, CPMV-specific RNA polymerase activity dependent on exogenous added template RNA could not be detected in extracts of these protoplasts in assays using poly(A)' oligo(U) or other template/primer combinations. In contrast, extracts of protoplasts in which poliovirus polymerase was produced exhibited RNA polymerase activity in such assays. These results indicate that the CPMV polymerase, unlike the poliovirus polymerase, is not able to use oligo(U) as a primer or cannot function on exogenous template and primer RNA.

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“…Determination of RNA polymerase activity in these extracts was as described previously (Flanegan & Baltimore, 1977;van Bokhoven et al, 1991).…”

Section: Methodsmentioning

confidence: 99%

Protoplasts transiently expressing the 200K coding sequence of cowpea mosaic virus B-RNA support replication of M-RNA

Bokhoven

Verver

Wellink

et al. 1993

Journal of General Virology

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“…To the extent that these activities in plants or at least their sometimes very great increase was attributed to a virus, they were expected to be more or less specific in requiring that viral RNA as template. This is the case for the bacterial and animal RNA virus replicases (19,20). However, the plant virologists were uniformly disappointed, in that their solubilized enzymes would accept any RNA and many polynucleotides as templates.…”

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confidence: 99%

“…This virus resembles the animal picornaviruses in many regards and has even more RNA than these viruses, which definitely code for their specific RNA polymerase (20,23). Without doubt viral genes are involved in eliciting the increased production ofplant enzymes, and in two divided-genome viruses we know that these genes are on the larger RNA [more than 2 x 106 daltons in cowpea mosaic virus (24) and tobacco rattle virus (25)].…”

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confidence: 99%

RNA-dependent RNA polymerases of plants

Fraenkel‐Conrat

1983

Proc. Natl. Acad. Sci. U.S.A.

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The existence of RNA-dependent RNA polymerases (EC 2.7.7.48) in plants has been definitely proven by their isolation in pure form from cucumber and tobacco in our laboratory and from cowpea at Wageningen. These enzymes are single-chain proteins of 100-130 kilodaltons. They show clear physical and biochemical differences characteristic for a given plant species, even when their amounts in the plants were greatly increased prior to isolation by infection with the same virus. The role of these enzymes in plant physiology remains unknown. A few years later several laboratories took up the study ofthe RNA-dependent RNA polymerase in tobacco, and they showed that the properties ofstill very crude enzyme preparations were indistinguishable from those of the enzyme present in greater amounts in plants infected with tobacco necrosis virus or alfalfa mosaic virus (3-6). Biochemists and molecular biologists, however, have shown scant interest in the existence of this class of enzymes of eukaryotic cells. Thus we continue to see books, chapters, and papers entitled "RNA polymerases" with the tacit assumption that these are all DNA transcriptases.Because the amounts of these enzymes in plants are usually low, but increased by RNA virus infection, interest in them has remained mostly restricted to plant virologists. Among these there were several camps holding at different times different opinions. One small group for a while retained doubts that such enzymes really existed in their uninfected control plants. Ac-tually in one plant, cucumber, no definite evidence for this enzyme has been reported. Another group claimed that the virusspecific enzymes (the viral RNA replicases) differed from the plant RNA-dependent RNA polymerases. As stated, virus infection is often used to increase the amounts and thus facilitate the purification and biochemical study of these enzymes. The fact that in association with viral RNA they are largely membrane bound, whereas in healthy plants they are mostly soluble or cytoplasmic, has represented an obstacle in the realization that these were nevertheless the same enzymes. Our conviction (3, 5) that they differed only in amount and localization has, however, gained ground in recent years. Data published since 1978, as well as data presented at recent international congresses, have illustrated this evolution of increased harmony (7-12,*).The progress in this research and the consensus arrived at in the past few years will now be briefly reviewed, followed by a consideration of the important questions: how do viruses increase the amounts of these enzymes, and what is their physiological role in the plants?The successful isolation ofa pure plant RNA-dependent RNA polymerase from healthy cauliflower was reported in the second and very brief paper by (15)(16)(17)(18), showed that various properties of the enzyme from tobacco and cowpea differed, even when the plants were infected by the same virus. This proved that the enzyme upon virus infection was still completely host-specific, thus not ...

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“…Purified FMDV RNA polymerase is capable of initiating RNA synthesis in vitro on an FMDV RNA template. The enzyme also tran-scribes in vitro the synthetic template poly(A) primed with (Up)5U with a poly(U) polymerase activity (6). Biochemical mapping of FMDV-coded proteins with pactamycin has shown that P56a is encoded by a region close to the 3' end of the genome (7,8).…”

mentioning

confidence: 99%

Temperature-sensitive RNA polymerase mutants of a picornavirus.

Lowe¹,

King²,

McCahon³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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Temperature-sensitive (ts) RNA polymerase mutants of a picornavirus are reported, Two foot-and-mouth disease virus (FMDV) mutants designated ts 22 and to 115 have been characterized. As judged by isoelectric focusing, both have charge alterations in P56a, the FMDV RNA polymerase protein. Virus RNA synthesis in cells infected with the mutants is severely impaired at the nonpermissive temperature. RNA polymerase purified from baby hamster kidney cells infected with these mutants exhibits a marked to transcribing activity in vitro. Spontaneous revertants of both mutants have P56a polypeptides that are indistinguishable from the parental proteins on the basis of charge. The revertants regain the ability to synthesize virus RNA in vivo at the nonpermissive temperature. RNA polymerase purified from the revertants remains transcriptionally active at the nonpermissive temperature.The genome of foot-and-mouth disease virus (FMDV) is a single-stranded "positive"-sense RNA molecule of approximately 8 kilobases with a small protein termed VPg covalently attached to its 5' end, an internal poly(C) tract, and a poly(A) sequence at the 3' end (1) (Fig. 1) (14,15). Hence, a tentative alignment of the genetic and biochemical maps has been constructed (15). We describe how a modified EF technique was used to screen mutants with ts loci at the 3' end of the FMDV genetic map for an altered P56a polypeptide. Two candidate RNA polymerase mutants (ts 22 and.ts 115) were identified by these criteria. Cells infected with these mutants synthesize only trace amounts of virus RNA at the nonpermissive temperature. Furthermore, FMDV RNA polymerase purified from the cytoplasm of baby hamster kidney cells infected with the mutants showed a ts transcribing activity in vitro when compared with the enzyme extracted from cells infected with the parental virus. MATERIALS AND METHODSViruses. The parental FMDV strain from which all mutants were derived was 0 Pacheco (ts+). Mutants mapping at the 3' end of the FMDV genome (5-fluorouracil-induced ts 22 and spontaneous ts 115) have been described (12, 13). The isolation ofa revertant ofts 22 (designated ts 22R) also has been described (15 The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Poliovirus-specific primer-dependent RNA polymerase able to copy poly(A).

Cited by 152 publications

References 21 publications

Protoplasts transiently expressing the 200K coding sequence of cowpea mosaic virus B-RNA support replication of M-RNA

Protoplasts transiently expressing the 200K coding sequence of cowpea mosaic virus B-RNA support replication of M-RNA

RNA-dependent RNA polymerases of plants

Temperature-sensitive RNA polymerase mutants of a picornavirus.

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